Method and composition for disease diagnosis according to saccharide binding

ABSTRACT

Disclosed is a method for characterizing carbohydrate polymer by identifying at least two binding agents that bind to the carbohydrate polymer. Binding is preferably determined by contacting the carbohydrate polymer with substrate that contains a plurality of first saccharide-binding agents affixed at predetermined locations on the substrate. The carbohydrate polymer is allowed to contact the substrate under conditions that allow for formation of a first complex between the first saccharide-binding agent and the carbohydrate polymer. A second saccharide-binding agent, which preferably includes a label, is also contacted with the carbohydrate polymer under conditions that allow for formation of a second complex between the second binding agent and the first complex. Identification of the first and second binding agent allows for characterization of the polysaccharide.

FIELD OF THE INVENTION

The invention relates generally to a method for analyzing moleculescontaining polysaccharides and more particularly to a method foranalyzing polysaccharides based using saccharide-binding agents such aslectins.

BACKGROUND OF THE INVENTION

Polysaccharides are polymers that include monosaccharide (sugar) unitsconnected to each other via glycosidic bonds. These polymers have astructure that can be described in terms of the linear sequence of themonosaccharide subunits, which is known as the two-dimensional structureof the polysaccharide. Polysaccharides can also be described in terms ofthe structures formed in space by their component monosaccharidesubunits.

A chain of monosaccharides that form a polysaccharide has two dissimilarends. One end contains an aldehyde group and is known as the reducingend. The other end is known as the non-reducing end. A polysaccharidechain may also be connected to any of the C1, C2, C3, C4, or C6 atom ifthe sugar unit it is connected to is a hexose. In addition, a givenmonosaccharide may be linked to more than two different monosaccharides.Moreover, the connection to the C1 atom may be in either the α or βconfiguration. Thus, both the two-dimensional and three-dimensionalstructure of the carbohydrate polymer can be highly complex.

The structural determination of polysaccharides is of fundamentalimportance for the development of glycobiology. Research in glycobiologyrelates to subjects as diverse as the identification andcharacterization of antibiotic agents that affect bacterial cell wallsynthesis, blood glycans, growth factor and cell surface receptorstructures involved in viral disease, and autoimmune diseases such asinsulin dependent diabetes, rheumatoid arthritis, and abnormal cellgrowth, such as that which occurs in cancer.

Polysaccharides have also been used in the development of biomaterialsfor contact lenses, artificial skin, and prosthetic devices.Furthermore, polysaccharides are used in a number of non-medical fields,such as the paper industry. Additionally, of course, the food and drugindustry uses large amounts of various polysaccharides andoligosaccharides.

In all of the above fields, there is a need for improved saccharideanalysis technologies. Saccharide analysis information is useful in,e.g, for quality control, structure determination in research, and forconducting structure-function analyses.

The structural complexity of polysaccharides has hindered theiranalysis. For example, saccharides are believed to be synthesized in atemplate-independent mechanism. In the absence of structuralinformation, the researcher must therefore assume that the buildingunits are selected from any of the saccharide units known today. Inaddition, these units may have been modified, during synthesis, e.g., bythe addition of sulfate groups.

Second, saccharide can be connected at any of the carbon moieties, e.g.,a the C1, C2, C3, C4, or C6 atom if the sugar unit it is connected to isa hexose. Moreover, the connection to the C1 atom may be in either α orβ configuration.

Third, saccharides may be branched, which further complicates theirstructure and the number of possible structures that have an identicalnumber and kind of sugar units.

A fourth difficulty is presented by the fact that the difference instructure between many sugars is minute, as a sugar unit may differ fromanother merely by the position of the hydroxyl groups (epimers).

The use of a plurality of such saccharide-binding agents, whether fixedto the substrate and/or employed as the second (soluble)saccharide-binding agent, characterizes the carbohydrate polymer ofinterest by providing a “fingerprint” of the saccharide. Such afingerprint can then be analyzed in order to obtain more informationabout the carbohydrate polymer. Unfortunately, the process ofcharacterization and interpretation of the data for carbohydrate polymerfingerprints is far more complex than for other biological polymers,such as DNA for example. Unlike binding DNA probes to a sample of DNAfor the purpose of characterization, the carbohydrate polymerfingerprint is not necessarily a direct indication of the components ofthe carbohydrate polymer itself. DNA probe binding provides relativelydirect information about the sequence of the DNA sample itself, sinceunder the proper conditions, recognition and binding of a probe to DNAis a fairly straightforward process. Thus, a DNA “fingerprint” which isobtained from probe binding can yield direct information about theactual sequence of DNA in the sample.

By contrast, binding of agents to carbohydrate polymers is not nearly sostraightforward. As previously described, even the two-dimensionalstructure (sequence) of carbohydrate polymers is more complex than thatof DNA, since carbohydrate polymers can be branched. These branchesclearly affect the three-dimensional structure of the polymer, and hencethe structure of the recognition site for the binding agent. Inaddition, recognition of binding epitopes on carbohydrate polymers bythe binding agents may be affected by the “neighborhood” of the portionof the molecule which is surrounding the epitope. Thus, the analysis ofsuch “fingerprint” data for the binding of agents to the carbohydratepolymer of interest is clearly more difficult than for DNA probebinding, for example.

A useful solution to this problem would enable the fingerprint data tobe analyzed in order to characterize the carbohydrate polymer. Such ananalysis would need to transform the raw data, obtained from thepreviously described process of incubating saccharide-binding agentswith the carbohydrate polymer, into a fingerprint which would itselfcontain information. The fingerprint would also need to be standardizedfor comparison across different sets of experimental conditions and fordifferent types of saccharide-binding agents. Unfortunately, such asolution is not currently available.

In spite of these difficulties, a number of methods for the structuralanalysis of saccharides have been developed. For example, PCTApplication No. WO 93/24503 discloses a method wherein monosaccharideunits are sequentially removed from the reducing end of anoligosaccharide by converting the monosaccharide at the reducing end toits keto- or aldehyde form, and then cleaving the glycosidic bondbetween the monosaccharide and the next monosaccharide in theoligosaccharide chain by using hydrazine. The free monosaccharides areseparated from the oligosaccharide chain and identified bychromatographic methods. The process is then repeated until allmonosaccharides have been cleaved.

PCT Application No. WO 93/22678 discloses a method of sequencing anunknown oligosaccharide by making assumptions upon the basic structurethereof, and then choosing from a number of sequencing tools (such asglycosidases) one which is predicted to give the highest amount ofstructural information. This method requires some basic information asto the oligosaccharide structure (usually the monosaccharidecomposition). The method also illustrates the fact that reactions withsequencing reagents are expensive and time-consuming, and thereforethere is a need for a method that reduces these expenses.

PCT Application No. WO 93/22678 discloses a method for detectingmolecules by probing a monolithic array of probes, such asoligodeoxynucleotides, immobilized on a VLSI chip. This publicationteaches that a large number of probes can be bound to an immobilizedsurface, and the reaction thereof with an analyte detected by a varietyof methods, using logic circuitry on the VLSI chip.

European Patent Application No. EP 421,972 discloses a method forsequencing oligosaccharides by labeling one end thereof, dividing thelabeled oligosaccharide into aliquots, and treating each aliquot with adifferent reagent mix (e.g. of glycosidases), pooling the differentreaction mixes, and then analyzing the reaction products, using,chromatographic methods. This method is useful for N-linked glycansonly, as they have a common structure at the point where the saccharidechain is linked to the protein. O-linked glycans are more varied, andthe method has as yet not been adapted for such oligosaccharides withgreater variability in their basic structure.

There is therefore a need for a system and method for characterizingpolysaccharides using an accurate, high throughput method foridentifying agents that bind to the polysaccharide.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery of a method for quicklyand accurately identifying agents that bind a given carbohydratepolymer. Also provided by the invention is a method for generating afingerprint of a carbohydrate polymer that is based on its pattern ofbinding to saccharide-binding agents.

In one aspect, the invention features a method for characterizing acarbohydrate polymer. The carbohydrate polymer is contacted with asurface that includes at least one first saccharide-binding agentattached to a predetermined location on the surface under conditionsallowing for the formation of a first complex between the firstsaccharide-binding agent and the carbohydrate polymer. The surface isthen contacted with at least one second saccharide-binding agent underconditions allowing for formation of a second complex between the firstcomplex and the second saccharide-binding agent. The firstsaccharide-binding agent and second saccharide-binding agent are thenidentified, thereby characterizing the carbohydrate polymer.

Also provided by the invention is a method of generating a fingerprintof a carbohydrate polymer by contacting a carbohydrate polymer with afirst saccharide-binding agent, determining whether the carbohydratepolymer binds to the saccharide-binding reagent, contacting thecarbohydrate polymer with a second saccharide-binding agent, anddetermining whether the carbohydrate polymer binds to the secondsaccharide-binding reagent. Identification of the first and secondsaccharide-binding agent is used to generate a fingerprint of thecarbohydrate polymer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the Glycomolecule identity (GMID) cardsobtained for pasteurized goat's milk (A and B), non-pasteurized goat'smilk (C and D) and bovine milk (E).

FIG. 2 is a reproduction of the GMID cards obtained for variouslipopolysaccharide samples. Cards A to E correspond to LPS# 1, 7, 10, 15and 16 respectively.

FIG. 3 is a high-level logic flowchart that illustrates an algorithm forchoosing a set of colored lectins.

FIG. 4 is a flowchart of an exemplary method according to the presentinvention for performing a fingerprint assay with a GMID card.

DETAILED DESCRIPTION OF THE INVENTION

Provided by the invention is a method for characterizing a carbohydratepolymer by systemically assembling a representation of information thatdescribes the binding status of the carbohydrate polymer with respect tosaccharide-binding agents.

To assess binding, status, the carbohydrate polymer is added to asurface that includes at least one saccharide-binding agent attached toa predetermined location on the surface. The carbohydrate polymer isincubated with the surface under conditions allowing for the formationof a complex between the first saccharide-binding agent and thecarbohydrate polymer. The surface can then be washed if desired toremove unbound carbohydrate polymer. The surface is then contacted witha second saccharide-binding agent under conditions allowing forformation of a second complex between the first complex and the secondsaccharide-binding agent. The second agent preferably carries adetectable label to allow for detection of the second complex. Detectionof the second complex at a location on the substrate corresponding tothe location of a predetermined binding-agent allows for theidentification of the first and second binding agents as agents thatbind to the carbohydrate polymer. Detecting the first and second bindingagents provides structural information about the carbohydrate polymer.

While the method has been described by first contacting, thecarbohydrate polymer with the surface and then adding a detectablelabel, it is understood that this order is not obligatory. Thus, in someembodiments, the second agent is mixed with the carbohydrate polymer,and this complex is added to the surface.

In some embodiments, a plurality of saccharide-binding agents areattached to the surface. Similarly, a plurality of second detectablesaccharide-binding agents may be used. In preferred embodiments, aplurality of both first and second saccharide-binding agents are used.

Thus, in various embodiments, at least, 5, 10, 15, 25, 30, or 50 or morefirst saccharide-binding agents are attached to the surface. Preferably,each the first saccharide-binding agents are attached at spatiallydistinct regions of the substrate.

In other embodiments, at least 5, 10, 15, 25, 30, or 50 of moresecond-saccharide binding agents are used. Preferably, each of thesecond-saccharide have attached thereto distinguishable labels, i.e.,labels that distinguish one-second saccharide-binding agent from anothersecond saccharide-binding agent.

As used herein, a “carbohydrate polymer” includes any molecule with apolysaccharide component. Examples include polysaccharide, aglycoprotein, and glycolipid. While a carbohydrate polymer includes anysaccharide molecule containing two or more linked monosaccharideresidues, it is understood that in most embodiments, the carbohydratepolymer will include 10, 25, 50, 1000, or 10,000 or more monosaccharideunits. If desired, the carbohydrate polymer can be added to the surfaceafter digestion with a saccharide-cleaving agent. Alternatively, thecarbohydrate polymer can be added to the surface, allowed to bind to afirst saccharide-binding agent attached to the surface, and thendigested with a saccharide-cleaving agent.

In general, any agent that binds to a polysaccharide can be used as thefirst or second saccharide-binding agent. As is known in the art, anumber of agents that bind to saccharides have been described. One classof agents is the lectins. Many of these proteins bind specifically to acertain short oligosaccharide sequence. A second class of agents is anantibody that specifically recognize saccharide structures. A thirdclass of saccharide-binding agents is proteins that bind to carbohydrateresidues. For example, glycosidases are enzymes that cleave glycosidicbonds within the saccharide chain. Some glycosidases may recognizecertain oligosaccharide sequences specifically. Another class of enzymesis glycosyltransferases, which cleave the saccharide chain, but furthertransfer a sugar unit to one of the newly created ends.

For the purpose of this application, the term “lectin” also encompassessaccharide-binding proteins from animal species (e.g. “mammalianlectins”). Thus, carbohydrate polymers, like DNA or proteins, clearlyhave an important biological function which should be studied in greaterdetail.

A saccharide-binding agent is preferably an essentiallysequence-specific agent. As used herein, “essentially sequence-specificagent” means an agent capable of binding to a saccharide. The binding isusually sequence-specific, i.e., the agent will bind a certain sequenceof monosaccharide units only. However, this sequence specificity may notbe absolute, as the agent may bind other related sequences (such asmonosaccharide sequences wherein one or more of the saccharides havebeen deleted, changed or inserted). The agent may also bind, in additionto a given sequence of monosaccharides, one or more unrelated sequences,or monosaccharides.

The essentially sequence-specific agent is usually a protein, such as alectin, a saccharide-specific antibody or a glycosidase orglycosyltransferase.

Examples of saccharide-binding agents lectins include lectins isolatedfrom the following plants: Conavalia ensiformis, Anguilla anguilla,Triticum vulgaris, Datura stramoniuim, Galanthus nivalis, Maackiaamurensis, Arachis hypogaea, Sambucus nigra, Erythrina cristagalli, Lensculinaris, Glycine may, Phaseolus vulgaris, Allomyrina dichotoma,Dolichos biflorus, Lotus tetragonolobus, Ulex europaeus, and Ricinuscommunis.

Other biologically active carbohydrate-binding compounds includecytokines, chemokines and growth factors. These compounds are alsoconsidered to be lectins for this patent application.

Examples of glycosidases include α-Galactosidase, β-Galactosidase,N-acetylhexosaminidase, α-Mannosidase, β-Mannosidase, α-Fucosidase, andthe like. Some of these enzymes may, depending upon the source ofisolation thereof, have a different specificity. The above enzymes arecommercially available, e.g., from Oxford Glycosystems Ltd., Abingdon,OX14 1RG, UK, Sigma Chemical Co., St. Lois, Mo., USA, or Pierce, POB.117, Rockford, 61105 USA.

The saccharide-binding agent can also be a cleaving agent. A “cleavingagent” is an essentially sequence-specific agent that cleaves thesaccharide chain at its recognition sequence. Typical cleaving agentsare glycosidases, including exo- and endoglycosidases, andglycosyltransferases. However, chemical reagents capable of cleaving aglycosidic bond may also serve as cleaving agents, as long, as they areessentially sequence-specific. The term “cleaving agent” or “cleavageagent” is within the context of this specification synonymous with theterm “essentially sequence-specific agent capable of cleaving”.

The cleaving agent may act at a recognition sequence. A “recognitionsequence” as used herein is the sequence of monosaccharides recognizedby an essentially sequence-specific agent. Recognition sequences usuallycomprise 2-4 monosaccharide units. An example of a recognition sequenceis Galβ1-3 GalNAc, which is recognized by a lectin purified from Arachishypogaea. Single monosaccharides, when specifically recognized by anessentially sequence-specific agent, may, for the purpose of thisdisclosure, be defined as recognition sequences.

The reaction conditions for the various essentially sequence-specificagents are known in the art. Alternatively, the skilled person mayeasily perform a series of tests with each essentially sequence-specificagent, measuring the binding activity thereof, under various reactionconditions. Advantageously, knowledge of reaction conditions under whicha certain essentially sequence-specific agent will react, and ofconditions under which it remain inactive, may be used to controlreactions in which several essentially sequence-specific reagents arepresent. For example, the second and third sequence-specific reagentsmay be added to the reaction simultaneously, but via a change inreaction conditions, only the second essentially sequence-specific agentmay be allowed to be active. A further change in reaction conditions maythen be selected in order to inactivate the second essentiallysequence-specific agent and activate the third essentiallysequence-specific agent. Some illustrative examples of reactionconditions are listed in the Table 1 below. In addition to the pH andtemperature data listed in Table 1, other factor, e.g. the presence ofmetals such as Zn, or salts of cations such as Mn, Ca, Na, such assodium chloride salt, may be investigated to find optimum reactionconditions or conditions under which certain essentiallysequence-specific agent will be active while others are inactive.

TABLE 1 Reaction conditions for some essentially sequence-specificagents codes for Condition condition serial Temp sets number pH (C.)Enzyme(s)

 ♡ 1 3.5 30 Jackbean β- galactosidase ♡ 2 5.0 37 Endo a-NAcetylgalactosidase α 1,2 Fucosidase β1,2 galactosidase

 

3 5.0 25 Bovine kidney α Fucosidase ♡ 

4 7.2 25 Coffee bean α galactosidase

 ♡ 

5 5.8 55 B. Fragilis Endo β-galactosidase 6 6.2 25 Chicken egg lysozyme7 4.3 37 Bovine testes β 1-3,4,6, Galactosidase from   2-9.5 50 Gly001-02 Biodiversa from 3.0-8.0 50 Gly 001-04 Biodiversa from  2-11 50Gly 001-06 BiodiversaSymbols represent enzyme groups which are separable by externalconditions. Diversa Corp. produces Thermophilic Endo/Exo glycosidaseswith a wide variety of activity in various pH and Temperatures

The first saccharide-binding agent may be immobilized using anyart-recognized method. For example, immobilization may utilizefunctional groups of the protein, such as amino, carboxy, hydroxyl, orthiol groups. For instance, a glass support may be functionalized withan epode group by reaction with epoxy silane, as described in the abovePCT publication. The epode group reacts with amino groups such as thefree ε-amino groups of lysine residues. Another mechanism consists incovering a surface with electrometer materials such as gold, as alsodescribed in the PCT publication. As such materials form stableconjugates with thiol groups, a protein may be linked to such materialsdirectly by free thiol groups of cysteine residues. Alternatively, thiolgroups may be introduced into the protein by conventional chemistry, orby reaction with a molecule that contains one or more thiol groups and agroup reacting with free amino groups, such as the N-hydroxylsuccinimidyl ester of cysteine. Also thiol-cleavable cross-linkers, suchas dithiobis(succinimidyl propionate) may be reacted with amino groupsof a protein. A reduction with sulfhydryl agent will then expose freethiol groups of the cross-linker.

The label attached to the second detectable label can be any label thatis detected, or is capable of being detected. Examples of suitablelabels include, e.g., chromogenic label, a radiolabel, a fluorescentlabel, and a biotinylated label. Thus, the label can be, e.g., coloredlectins, fluorescent lectins, biotin-labeled lectins, fluorescentlabels, fluorescent antibodies, biotin-labeled antibodies, andenzyme-labeled antibodies. In preferred embodiments, the label is achromogenic label. The term “chromogenic binding agent” as used hereinincludes all agents that bind to saccharides and which have a distinctcolor or otherwise detectable marker, such that following binding to asaccharide, the saccharide acquires the color or other marker. Inaddition to chemical structures having intrinsic, readily-observablecolors in the visible range, other markers used include fluorescentgroups, biotin tags, enzymes (that may be used in a reaction thatresults in the formation of a colored product), magnetic and isotopicmarkers, and so on. The foregoing list of detectable markers is forillustrative purposes only, and is in no way intended to be limiting orexhaustive. In a similar vein, the term “color” as used herein (e.g. inthe context of step (e) of the above described method) also includes anydetectable marker.

The label may be attached to the second saccharide-binding agent usingmethods known in the art. Labels include any detectable group attachedto the saccharide or essentially sequence-specific agent that does notinterfere with its function. Labels may be enzymes, such as peroxidaseand phosphatase. In principle, also enzymes such as glucose oxidase andβ-galactosidase could be used. It must then be taken into account thatthe saccharide may be modified if it contains the monosaccharide unitsthat react with such enzymes. Further labels that may be used includefluorescent labels, such as Fluorescein, Texas Red, Lucifer Yellow,Rhodamine, Nile-red, tetramethyl-rhodamine-5-isothiocyanate,1,6-diphenyl-1,3,5-hexatriene, cis-Parinaric acid, Phycoerythrin,Allophycocyanin, 4′,6-diamidino-2-phenylindole (DAPI), Hoechst 33258,2-aminobenzamide, and the like. Further labels include electron densemetals, such as gold, ligands, haptens, such as biotin, radioactivelabels.

The second saccharide-binding agent can be detected using enzymaticlabels. The detection of enzymatic labels is well known in the art ofELISA and other techniques where enzymatic detection is routinely used.The enzymes are available commercially, e.g., from companies such asPierce.

In some embodiments, the label is detected using, fluorescent labels.Fluorescent labels require an excitation at a certain wavelength anddetection at a different wavelength. The methods for fluorescentdetection are well known in the art and have been published in manyarticles and textbooks. A selection of publications on this topic can befound at p. O-124 to O-126 in the 1994 catalog of Pierce. Fluorescentlabels are commercially available from Companies such as SIGMA, or theabove-noted Pierce catalog.

The second saccharide-binding agent may itself contain a carbohydratemoiety and/or protein. Coupling labels to proteins and sugars aretechniques well known in the art. For instance, commercial kits forlabeling saccharides with fluorescent or radioactive labels areavailable from Oxford Glycosystems, Abingdon, UK. Reagents andinstructions for their use for labeling proteins are available from theabove-noted Pierce catalog.

Coupling is usually carried out by using functional groups, such ashydroxyl, aldehyde, keto, amino, sulfhydryl, carboxylic acid, or thelike groups. A number of labels, such as fluorescent labels, arecommercially available that react with these groups. In addition,bifunctional cross-linkers that react with the label on one side andwith the protein or saccharide on the other may be employed. The use ofcross-linkers may be advantageous in order to avoid loss of function ofthe protein or saccharide.

The label can be detected using methods known in the art. Some detectionmethods are described in the above-noted WO 93/22678, the disclosure ofwhich is incorporated herein in its entirety. Particularly suitable forthe method of the present invention is the CCD detector method,described in the publication. This method may be used in combinationwith labels that absorb light at certain frequencies, and so block thepath of a test light source to the VLSI surface, so that the CCD sensorsdetect a diminished light quantity in the area where the labeled agenthas bound. The method may also be used with fluorescent labels, makinguse of the fact that such labels absorb light at the excitationfrequency. Alternatively, the CCD sensors may be used to detect theemission of the fluorescent label, after excitation. Separation of theemission signal from the excitation light may be achieved either byusing sensors with different sensitivities for the differentwavelengths, or by temporal resolution, or a combination of both.

In some embodiments, the method further includes acquiring one or moreimages of the first saccharide-binding agent and the saccharide-bindingagent. The information can be is stored, e.g., as a photograph ordigitized image. Alternatively, the information provided by the firstand second binding image can be stored in a database.

The invention also includes a substrate that includes a plurality ofcomplexes. Each complex includes a first saccharide-binding agent boundto a predetermined location on the substrate. The substrate can alsooptionally include a saccharide bound to the first saccharide-bindingagent and/or a detectable second saccharide-binding agent. In someembodiments, the substrate is provided in the form of a solid supportthat includes in a pre-defined order a plurality of visual or otherwisedetectable markers representative of a saccharide or saccharide sequenceor fragment.

If desired, a substrate containing a plurality of firstsaccharide-binding agents can be provided in the form of a kit.Diagnostic procedures using the methods of this invention may beperformed by diagnostic laboratories, experimental laboratories,practitioners, or private individuals. This invention providesdiagnostic kits which can be used in these settings. The presence orabsence of a particular carbohydrate polymer, as revealed by its patternof reacting with saccharide binding agent, may be manifest in a providesample. The sample can be, e.g., clinical sample obtained from that anindividual or other sample.

Each kit necessarily comprises saccharide-binding agent or agents whichrenders the procedure specific. The reagent is preferably supplied in asolid form or liquid buffer that is suitable for inventory storage, andlater for exchange or addition into the reaction medium when the test isperformed. Suitable packaging is provided. The kit may optionallyprovide additional components that are useful in the procedure. Theseoptional components include buffers, capture reagents, developingreagents, labels, reacting surfaces, means for detection, controlsamples, instructions, and interpretive information.

The kit may optionally include a detectable second saccharide-bindingagent and if desired, reagents of detecting the second binding agent.The plurality of first saccharide-binding agents are preferably attachedat predetermined location on the substrate and a detectable secondsaccharide-binding agent. In other embodiments, the kit is provided witha substrate and first saccharide-binding agents that can be attached tothe substrate, as well as second saccharide-binding agents.

Generating Fingerprints of Carbohydrate Polymers

The method and reagents described above can be used to generate afingerprint of a carbohydrate polymer. As used herein, a fingerprint ofa carbohydrate polymer is a compilation of information about the bindingstatus of the carbohydrate polymer and a plurality of scattered-bindingagents. In some embodiments, the fingerprint is a numeric representationof the detection of the presence of binding by the saccharide-bindingagents to the carbohydrate polymer.

The fingerprint of the carbohydrate polymer can be generated bycontacting the carbohydrate polymer with a first saccharide-bindingagent and determining whether the carbohydrate polymer binds to thesaccharide-binding reagent. The carbohydrate polymer is also contactedwith a second saccharide-binding agent, and a determination is made asto whether the second binding-agent binds to the carbohydrate polymer.

The carbohydrate polymer is preferably contacted with at least fivesaccharide-binding agents, and a determination is made as to whether thecarbohydrate polymer binds to each of the at least fivesaccharide-binding reagents. In preferred embodiments, the binding ofthe carbohydrate polymer to at least 10, 15, 20, or 25 or more agents isdetermined.

In preferred embodiments, binding of the first and secondsaccharide-agent is determined by providing a surface comprising atleast one first saccharide-binding agent attached to a predeterminedlocation on the surface and contacting the surface with a carbohydratepolymer under conditions allowing for the formation of a first complexbetween the first saccharide-binding agent and the carbohydrate polymer.Unbound polymer is removed if desired and the surface is contacted withat least one second saccharide-binding agent under conditions allowingfor formation of a second complex between the first complex and thesecond saccharide-binding agent. The first and second saccharide-bindingagent are then identified, and the information generated provides afingerprint for the carbohydrate polymer. By including a plurality offirst and/or second saccharide-binding agents, it is possible togenerate a detailed fingerprint of the carbohydrate polymer. Of course,it will be apparent to one of ordinary skill in the art that the absenceof binding of a first or second saccharide-agent to a carbohydratepolymer will also contribute to the fingerprint generated for thepolysaccharide.

The second saccharide agent preferably contains a detectable label. Whenthe second saccharide-binding agent is labeled, the identity of thesecond label determines the identity of the second saccharide-bindingagent. The position of the second label on the substrate in turn revealsthe identity of the first saccharide-binding agent.

The invention will be further illustrated in the following examples,which do not limit the scope of the appended claims.

Example 1 Glycomolecule Analysis Using Antibodies as First and SecondSequence-Specific Agents

This example further illustrates the technique of analyzingglycomolecules according to the invention. As a first and secondsequence-specific agent, antibodies are used. The following tables liststhe results of reactions with two different saccharides denoted forpurposes of illustration, HS and NS.

The structure of the sugars is as follows:

Table 2 lists the results of the reaction between the saccharide and thefirst and second essentially sequence-specific agents, which areantibodies against T-antigen, Lewis^(x) (Le^(x)), or Lewis^(b) antigen(Le^(b)). The first essentially sequence-specific agent is immobilizedon a matrix, preferably a solid phase microparticle. The secondessentially sequence-specific agent is labeled with a fluorescent agent,i.e., nile-red or green color. In addition, the reducing end of thesaccharide is labeled, using a label clearly distinguishable from therifle-red or green color label which act as markers for the secondessentially sequence-specific agents. Table 2 lists the reactions forthe saccharide HS, while Table 3 lists the reactions for the saccharideNS.

TABLE 2 On the matrix anti T-antigen Anti-Le^(X) anti-Le^(b) Saccharidebound HS HS Second mAb nile-red anti-Le^(X) Signal nile-red, Reducingnone reducing end end

TABLE 3 On the matrix anti T-antigen Anti-Le^(X) anti-Le^(b) Saccharidebound NS NS Second mAb Green anti-Le^(b) nile-red anti-Le^(X) SignalGreen, nile-red, reducing end reducing end

In summary, the following signals are now detectable in the reactions ofthe saccharide HS or NS (rows) when using the indicated antibodies asfirst essentially sequence-specific agent (columns):

TABLE 4 On the matrix anti T-antigen Anti-Le^(X) anti-Le^(b) HSnile-red, Reducing reducing end end NS Green, nile-red, reducing endreducing end NS Green, nile red, reducing end reducing end

After the label has been detected and the result recorded for eachreaction, a third essentially sequence-specific agent is added. In thisexample, two independent reactions with a third essentiallysequence-specific agent are used. The solid phase carrying the sugarmolecule may now be advantageously divided into aliquots, for reactionwith either α1-2 Fucosidase or Exo β galactosidase (third essentiallysequence-specific agents). Alternatively, three sets of reactions with afirst and second essentially sequence-specific agent may be carried out.

TABLE 5 reactions after applying α1-3,4 Fucosidase: On the matrix antiT-antigen Anti-Le^(X) anti-Le^(b) HS reducing end NS

TABLE 6 reaction after applying Exo β galactosidase from D. pneumoniae(EC 3.2.1.23 catalog number 1088718 from Boehringer Mannheim, 68298Mannheim, Germany) On the matrix anti T-antigen Anti-Le^(X) anti-Le^(b)HS nile-red NS Green nile-red

TABLE 7 reactions after applying α1-2 Fucosidase: On the matrix antiT-antigen Anti-Le^(X) anti-Le^(b) HS nile-red, Reducing end reducing endNS Reducing end

From the data gathered as explained above, a glycomolecule identity(GMID) card can now be created. An example for such information islisted in Table 8 for saccharide HS and in Table 9 for saccharide NS.

TABLE 8 On the matrix anti T-antigen Anti-Le^(X) anti-Le^(b) 0 nile-red,Reducing reducing end end 1 reducing end — — 2 nile-red 3 nile-red,Reducing reducing end end

TABLE 9 On the matrix anti T-antigen Anti-Le^(X) anti-Le^(b) 0 Green,nile red, reducing end reducing end 1 — — — 2 Green nile red 3 Reducingend

The identity of the second and third essentially sequence-specificagents need not be disclosed in such a data list. For the purpose ofcomparison, it is sufficient that a certain code number (1, 2 or 3 inthe above tables) always identifies a certain combination of reagents.

Example 2 A Scheme for the Sequential Labeling of Reducing Ends

As has been indicated in the description and example above, the methodof the invention advantageously uses labeling of the saccharide to beinvestigated at its reducing end. However, this labeling technique maybe extended to sites within the saccharide, and thus contribute to themethod of the invention, by providing more information. As it ispossible to label the saccharide within the chain, by cleavage using anendoglycosidase followed by labeling of the reducing end, it istherefore possible to obtain a labeled reducing end within thesaccharide chain. As that reducing end is necessarily closer to thebinding sites for the first, second and third essentiallysequence-specific agents, compared to the original reducing end, the useof an internally created labeled reducing end provides additionalinformation. Moreover, it is possible, by sequentially labeling ofreducing ends according to the method described further below, toidentify the sites for distinct glycosidases in sequential order on thechain of the saccharide to be investigated.

The method of sequential labeling of reducing ends is now described inmore detail in the following steps:

1. Blocking:

A polysaccharide having a reducing end is incubated in a solutioncontaining NaBH₄/NaOH at pH 11.5.

This treatment blocks the reducing end, so that the polysaccharide isnow devoid of a reducing end (RE).

2. Exposing:

The polysaccharide of step 1 is treated with an endoglycosidase. If therecognition site for that endoglycosidase is present within thepolysaccharide, a new reducing end will be created by cleavage of thepolysaccharide. The solution now contains two saccharides: the fragmentwith the newly exposed RE in the endoglycosidase site, and the secondfragment whose RE is blocked.

3: Labeling of the Reducing End

This reaction may be carried out using e.g., 2-aminobenzamide(commercially available in kit form for labeling saccharides by OxfordGlycosystems Inc., 1994 catalog, p. 62). After the reaction underconditions of high concentrations of hydrogen and in high temperature(H+/T), followed by reduction, has been completed, the mixture containstwo fragments, one of which is labeled at its reducing end, while theother remains unlabeled due to the fact that its reducing end isblocked.

Another way to label reducing ends is by reductive amination.Fluorescent compounds containing arylamine groups are reacted with thealdehyde functionality of the reducing end. The resulting CH═N doublebond is then reduced to a CH₂—N single bond, e.g., using sodiumborohydride. This technology is part of the FACE (Fluorophore assistedCarbohydrate Electrophoresis) kit available from Glyko Inc., Novato,Calif., USA, as detailed e.g., in the Glyko, Inc. catalog, p. 8-13,which is incorporated herein by reference.

4. Reaction with a Second Endoglycosidase

A second endoglycosidase may now be reacted with the saccharide mixture.The new reaction mixture has now three fragments, one with an intactreducing end, a second with a reducing end labeled by 2-aminobenzimide,and a third with a blocked reducing end.

Example 3 Derivation of Structural Information from a Series ofReactions with Essentially Sequence-Specific Agents

This example further illustrates the method of the invention, i.e., thegeneration of data related to the structure of the saccharide by using aset of reactions as described further above. The example furtherdemonstrates that sequence information can be deduced from said set ofreactions.

In some cases, the reagents used may not react exactly as predicted frompublished data, e.g. taken from catalogs. For instance, the lectinDatura stramonium agglutinin as described further below is listed in theSigma catalog as binding. GlcNac. However, in the reactions detailedfurther below, DSA is shown to bind to Coumarin 120-derivatized Glc(Glc-AMC). It appears that Glc-AMC acts like GlcNac for all purposes,because of the structural similarity between these compounds. Further,as apparent from the results below, the endogalactosidase used cleavesnot only at galactose residues, but also the bond connecting the Glc-AMCgroup to the rest of the saccharide.

It is apparent that the essentially sequence-specific agents used in thepractice of the invention may in some cases have fine specificities thatvary from the specificity of these agents given in published material,e.g., catalogs. Such reactions can quickly be identified by using themethod of the invention with saccharides of known structure. The resultsfound may then be compared with expected results, and the differenceswill allow the identification of variant specificities of theessentially sequence-specific agents used. Such variation from publisheddata in fine specificities of essentially sequence-specific agents maythen be stored for future analysis of unknown saccharides structuresusing these agents.

In the following, the method of the invention is illustrated using anend-labeled pentasaccharide and various lectins and glycosidases. Thepentasaccharide has the structureGal-β(1,4)[Fuc-α(1,3)]-GlcNAc-β(1,3)-Galβ(1,4)-Glc. The pentasaccharideis branched at The GlcNAc position having fucose and galactose bound toit in positions 3 and 4 respectively. The pentasaccharide is labeled atits reducing end (Glc) with Coumarin-120 (7-amino-4-methyl coumarin,available, e.g., from Sigma, catalog No. A 9891). The coupling reactionmay be carried out as described above for the labeling of reducing endsby using arylamine functionalities. Coumarin-120, when excited at 312 nmemits blue fluorescence. As first and second essentiallysequence-specific agents, Endo-β-Galactosidase KEG, Boehringer Mannheim)and Exo-1,3-Fucosidase (FD, New England Biolabs) are used. The reactionconditions for both reagents are as described in the NEB catalogue forExo-1,3-Fucosidase.

Three reactions were carried out. The first included Fucosidase (FD) andEndo-Galactosidase (EG), the second, FD only, and the third, EG only. Afourth reaction devoid of enzyme served as control.

In order to ascertain that the enzymes had digested the saccharide, thevarious reactions are size-separated using thin-layer chromatography(TLC).

After separation, the saccharides on the TLC plate may detected byexposing the plate to ultraviolet light. The results are shown in thefollowing illustration.

In reaction 4, no glycosidase was added, so the saccharide is intact andmoves only a small distance on the plate. The fragment of reaction 2 issecond in molecular weight, while the fragments of reactions 1 and 3appear to be equal. From these data, it can be concluded that thesequence of the glycosidase sites on the saccharide is FD-EG-reducingend (coumarin-label).

The above pentasaccharide is now tested by a set of reactions asdescribed further above. As first and second essentiallysequence-specific agents, lectins were used. The lectins (AnguillaAnguilla agglutinin (AAA), catalog No. L4141, Arachis Hypogaeaagglutinin (PNA), catalog No. L0881, Ricinus communis agglutinin (RCA I)catalog No. L9138, Lens Culinaris agglutinin (LCA) catalog No. L9267,Arabs Precatorius agglutinin, (APA), catalog No. L9758) are availablefrom Sigma. Lectins are also available from other companies. Forinstance, RCA I may be obtained from Pierce, catalog No. 39913. Lectinsare immobilized by blotting onto nitrocellulose filters.

The reaction buffer is phosphate-buffered saline (PBS) with 1 mM CaCland 1 mM MgCl. After binding of the lectins, the filter was blocked with1% BSA in reaction buffer. As controls, reactions without lectin andwith 10 μg BSA as immobilized protein were used.

The results of the reactions are indicated in Table 10. A plus indicatesthe presence of 312 nm fluorescence, which indicates the presence of thecoumarin-labeled reducing end. The numerals 1-4 in the table indicatereactions as defined above.

TABLE 10 AAA PNA LCA DSA RCA I 1 ++ 2 ++ ++ ++ 3 ++ 4 ++ ++ ++ ++

From the results as listed in Table 10 (reaction 4-control) it isevident that lectins AAA, PNA, DSA and RCA-I bind the saccharide.Therefore, Fucose, Gal(1-3)GlcNAc, GlcNAc, and Galactose/GalNAc must bepresent in the saccharide, as these are the respective saccharidestructures that are recognized by AAA, PNA, DSA and RCA-I. It is furtherevident that the above described glycosidases Fucosidase andEndo-β-Galactosidase recognize cleavage sequences in the saccharide.These sequences are Fuc (1-3/1-4) GlcNAc andGlcNAcβ(1-3)Galβ(1-3/4)Glc/GlcNAc, respectively.

It can further be deduced that both glycosidase sites are locatedbetween the fucose sugar and the reducing end, as said end is cleaved byeither glycosidase when AAA (which binds to fucose) is used asimmobilized lectin. The reaction with DSA, on the other hand, allows thededuction that either the GlcNAc monosaccharide is located between theglycosidase sites and the reducing end, or that Glc is directly bound tothe coumarin, as neither glycosidase cleaves off the reducing end whenDSA is used as immobilized agent.

Moreover, the reaction with PNA as immobilized agent shows that thereducing end is cleaved only if Endo-βGalactosidase is used (reactions 1and 3). This indicates that the Endo-PGalactosidase site is locatedbetween the site for PNA and the reducing end. On the other hand, theFucosidase site must be located between the PNA site and the other endof the saccharide.

When taking into account the above data, it is now possible to propose asequence of the saccharide as follows:

Fucα(1-3, 1-4)GlcNAc(1-3)Gal(1-4)Glc/GlcNAc

reducing end

The above experiment clearly demonstrates that the method of theinvention can yield a variety of data, including sequence information,based upon relatively few reactions. Some details in the sequenceinformation may not be complete, such as the (1-3) or (1-4) connectionbetween Fucose and GlcNAc in the above saccharide. Had themonosaccharide composition of the pentasaccharide been known, then theabove analysis would have yielded all of the details of saidpentasaccharide. Nevertheless, the information gained even in theabsence of the monosaccharide composition data is very precise comparedto prior art methods.

Example 4 Derivation of Partial or Complete Sequence Information

The method of the invention is suitable for automation. Thus, the stepsdescribed above, for example, in examples 1 to 3, may be carried outusing an automated system for mixing, aliquoting, reacting, anddetection. The data obtained by such an automated process may then befurther processed in order to “collapse” the mapping information topartial or complete sequence information. The method for such dataprocessing is described in further detail below.

After all data have been collected, a comparison is made betweendetection signals obtained from reactions prior to the addition ofglycosidase, to signals obtained after the addition (and reaction with)of glycosidase. Those signals that disappear after reaction withglycosidase are marked. This may advantageously be done by preparing alist of those signals, referred to hereinafter as a first list. Theidentity of two sites on the polysaccharide may now be established foreach such data entry. The position in the (optionally virtual) arrayindicates the first essentially sequence-specific agent. If a signal hasbeen detected before reaction with the glycosidase, the recognition sitefor that agent must exist in the polysaccharide. The disappearance of asignal, for instance, of the signal associated with the secondessentially sequence-specific agent, now indicates that the glycosidasecleaves between the recognition sites of the first and secondessentially sequence-specific agents. The sequence of recognition sitesis therefore (first essentially sequence-specificagent)-(glycosidase)-(second essentially sequence-specific agent). Ifthe signal for the reducing end is still present after digestion withthe glycosidase, then the relative order of the recognition sequenceswith respect to the reducing end can be established; otherwise, bothpossibilities (a-b-c and c-b-a) must be taken into account. For thepurpose of illustration, the term “recognition site of the firstessentially sequence-specific agent” shall be denoted in the following“first recognition site”, the term “recognition site for the secondessentially sequence-specific agent” shall be denoted “secondrecognition site”, and the term “recognition site for glycosidase” shallbe denoted “glycosidase”.

It is now possible to create a second list of triplets of recognitionsites of the above type (type 1 triplets):

(first recognition site)-(glycosidase)-(second recognition site).

Similarly, a third list can now be created relating to (optionallyvirtual) array locations where all signals remain after addition ofglycosidase (type 2 triplets):

(glycosidase)-(first recognition site)-(second recognition site)

Obviously, a sufficient number of triplets defines a molecule in termsof its sequence, i.e., there can only be one sequence of saccharidesthat will contain all of the triplets found. A lower number of tripletsmay be required when information on the length of the molecule isavailable. The number of required triplets may be even lower if thetotal sugar content of the molecule is known. Both saccharide molecularweight and total monosaccharide content may be derived from prior artmethods well known to the skilled person.

The process of obtaining sequence information, i.e., of collapsing thetriplets into a map of recognition sites, is described below.

The second and third lists of triplet recognition sites are evaluatedfor identity (three out of three recognition sites identical), highsimilarity (two out of three recognition sites identical), and lowsimilarity (one out of three recognition sites identical). For thepurposes of illustration, it is now assumed that the polysaccharide is alinear polysaccharide, such as, for example, the saccharide portion ofthe glycan heparin.

The above second and third lists are then used to prepare therefrom aset of lists of triplets wherein each list in said set of lists containstriplets that share the same glycosidase recognition sequence. Bycomparing all triplets containing a certain glycosidase recognitionsequence with all triplets containing a second glycosidase recognitionsequence, it is now possible to divide the polysaccharide sequence intofour areas, ranging from the first end of the molecule to glycosidase 1(fragment a), from glycosidase 1 to glycosidase 2 (fragment b), and fromglycosidase 2 to the second end of the molecule (fragment c):

<first end> <glycosidase 1> <glycosidase2> <second end>

Identical recognition sites within triplets of type 2 with differentglycosidase sites, wherein said recognition sites are located in thesame direction in relation to the respective glycosidase site, arecandidates for the location within either the area a or c, depending onsaid location. Identical recognition sites within triplets of type 2with different glycosidase sites, wherein said recognition sites arelocated in different directions (e.g., one in the direction of thereducing end, in the other triplet, in the direction of the non-reducingend), are candidates for the location within the area b, i.e., betweenthe two glycosidase sites.

Identical recognition sites within triplets of type 1 with differentglycosidase sites are candidates for the location of one of the first orsecond recognition sites in area a (or c), and the other of said firstor second recognition sites being located in the area c (or a). That is,if one of the first or second recognition sites is located in area a,then the other of said first or second recognition sites must be locatedin area b, and vice versa. None of the said first or second recognitionsites may be located in area b.

Identical recognition sites within triplets of type 1 with differentglycosidase sites, wherein a given recognition site is located in one ofthe triplets; in the direction of the reducing end and in the othertriplet, in the direction of the non-reducing, are candidates for thelocation of said recognition site within area b.

Having established the above positional relationships for a number ofrecognition sites within the triplets, the total of the recognitionsequences can now be arranged in a certain order using logicalreasoning. This stage is referred to as a sequence map. If a sufficientnumber of recognition sequences are arranged, the full sequence of thesaccharide may be derived therefrom. As the method does not determinethe molecular weight of the saccharide, the chain length is unknown.Therefore, if the degree of overlap between the various recognitionsites is insufficient, there may be regions in the sequence whereadditional saccharide units may be present. Such saccharide units may beundetected if they do not fall within a recognition site of any of theessentially sequence-specific agents used. However, the entire sequenceinformation may also be obtained in this case, by first obtaining themolecular weight of the saccharide, which indicates its chain length,and secondly its total monosaccharide content.

Another possibility of closing gaps in the sequence map is the method ofexample 2, wherein sequential degradation by glycosidase is employed toderive sequence information.

The existence of branching points in the saccharide may complicate themethod as outline above. One remedy to that is to use glycosidases toprepare fractions of the molecule, and analyze these partial structures.The extent of branching in such partial structures is obviously lowerthan in the entire molecule. In addition, reagents may be employed thatspecifically recognize branching points. Examples for such reagents aree.g., the antibodies employed in example 1 above. Each of theseantibodies binds a saccharide sequence that contains at least onebranching point. Moreover, certain enzymes and lectins are availablethat recognize branched saccharide structures. For instance, the enzymepullanase (EC 3.2.1.41) recognizes a branched structure. In addition,antibodies may be generated by using branched saccharide structures asantigens. Moreover, it is possible to generate peptides that bindcertain saccharide structures, including branched structures (see e.g.,Deng S J, MacKenzie C R, Sadowska J, Miehniewicz J, Young N M, Bundle DR, Narang; Selection of antibody single-chain variable fragments withimproved carbohydrate binding by phage display. J. Biol. Chem. 269,9533-38, 1994).

In addition, knowledge of the structure of existing carbohydrates willin many cases predict accurately the existence of branching points. Forinstance, N-linked glycans possess a limited number of structures, aslisted at p. 6 of the oxford Glycosystems catalog. These structuresrange from monoantennary to pentaantennary. The more complicatedstructures resemble simpler structures with additional saccharideresidues added. Therefore, if monoantennary structure is identified, itis possible to predict all of the branching points in a more complicatedstructure, simply by identifying the additional residues and comparingthese data with a library of N-linked glycan structures.

Moreover, it will often be possible by analyzing data gathered accordingto the method of the invention, to deduce the existence and location ofbranching points logically. For instance, if two recognition sites,denoted a and b, are located on different branches, then digesting witha glycosidase whose site is located between the reducing end and thebranching point will result in loss of the reducing end marker. Themarkers for both recognition sites a and b, however, will remain. If aglycosidase located between the branching point and recognition site ais used, then the marker for recognition site b and the reducing endmarker will be cleaved off. Not taking into account the possibility ofbranching points, this would indicate that the recognition site b islocated between the recognition site a and the reducing end. However, ifa glycosidase located between the recognition site b and the branchingpoint is used, the reducing end marker and recognition site a will becleaved off. Again, not taking into account the possibility ofbranching, this would indicate that recognition site a is locatedbetween the reducing end and recognition site b. These deductions areobviously incompatible with one another, and can only be resolved if oneassumes that recognition sites a and b are located on two differentbranches. The branching point is located between the recognition sites aand b and the first of the above glycosidases. The other aboveglycosidases used are located on a branch each, between the branchingpoint and the respective recognition site (a or b).

Therefore, when using agents that recognize branched structures in themethod of the invention, as essentially sequence-specific agents, it ispossible to derive information on the existence and location ofbranching points in the saccharide molecule. This information can thenbe used to construct sequence maps of each branch of the structure,yielding a sequence map of the entire branched structure. The gaps insuch a structure may then be closed as in the case of unbranchedsaccharides, according to the invention, i.e., by using additionalreactions, by digestion with glycosidases, whereby the regions of themolecule where gaps exist are specifically isolated for further analysisaccording to the method of the invention; and by sequential glycosidasedigestion as described further above.

In summary, a method for determining the sequence of a saccharide and/orfor mapping the structure of said saccharide according to the inventioncomprises the steps of

1. collecting triplets of type 1 and type 22. sorting said triplets according to similarity3. comparing triplets with different glycosidase recognition sites4. arranging the triplets in the order of occurrence on the saccharide5. arranging the glycosidase recognition sites6. checking the compatibility to the triplets7. arranging recognition sequences of glycosidases and of first andsecond essentially sequence-specific agents in a single file order8. translating the recognition sequences (sites) into polysaccharidesequence9. correcting “overlap” problems10. outputting a sequence11. checking against all available data

After the above step 5 has been carried out, a preliminary order ofglycosidase sites has been established. In step 6, it is now checked foreach triplet whether predictions based thereon are in agreement withthat order. Then, based on contradiction in the data, a new model isgenerated that fits the data of the triplet. This model is then testedagainst the data of all triplets. Furthermore, additional reactions maybe carried out, in order to extract additional vectorial informationregarding the recognition sites that involve said triplet.

After the above step 8, wherein the sequentially arranged recognitionsites are translated into a sequence of actual monosaccharide units, amodel of the saccharide sequence can be suggested. In order to test saidmodel, a number of questions needs to be answered. The first of theseis, what is the minimum sequence that would still have the same sequencemap? At this stage, information on molecular weight and monosaccharidecomposition, if available, is not taken into account. This approachmerely serves the creation of a sequence which incorporates all of theavailable data with as few as possible contradictions. In that respect,the second question to be answered is, does the minimum sequence stillagree with all of the data available at that point (excluding optionalmolecular weight and monosaccharide composition data)? The thirdquestion to be answered is, do other sequences exist that would fit thesequence map as established? In the affirmative, the additionalsequences may then be tested using the question: How does each sequencemodel agree with the triplet information, and with additional optionaldata, such as information on the molecular weight, monosaccharidecomposition, and model saccharide structures known from biology.

Finally, the sequence model that has been found to be best according tothe steps 1-10 described above, will then be tested against alltriplets, monosaccharide composition, prior knowledge on the molecularweight and structural composition of the saccharide, and predictionsfrom biologically existent similar structures. By such repeated testing,the contradictions between the available data and the sequence model areidentified, and if possible, the sequence model is adapted to betterrepresent the data.

Example 5 Glycomolecule Identity (GMID) Analysis of Milk Samples

The aim of this example is to demonstrate the application of the GMIDtechnique to the analysis and comparison of milk samples.

A. Membranes and Layer Lectins:

The supporting surface used in the experiments described hereinbelow isa nitrocellulose membrane. The membranes were prepared as follows:

1. Nitrocellulose membranes were cut out and their top surface markedout into an array of 9×6 squares (3 mm² each square). The membranes werethen placed on absorbent paper and the top left square of each onemarked with a pen.2. Lyophilized lectins were resuspended in water to a finalconcentration of 1 mg/ml. The resuspended lectins (and a controlsolution: 5% bovine serum albumin) were vortex mixed and 1 μl of eachsolution is added to one of the 28 squares on the blot, indicated byshading in the following illustrative representation of a typical blot:

The lectins used in this experiment are listed in Table 11.

TABLE 11 Lectin Manufacturer Cat. No. WGA Vector MK2000 SBA VectorMK2000 PNA Vector MK2000 DBA Vector MK2000 UEA I Vector MK2000 CON AVector MK2000 RCA I Vector MK2000 BSL I Vector MK3000 SJA Vector MK3000LCA Vector MK3000 Swga Vector MK3000 PHA-L Vector MK3000 PSA VectorMK3000 AAA — — PHA-E Vector MK3000 PNA Leuven LE-408 LCA Sigma L9267 DSASigma L2766 APA — WGA Leuven LE-429 Jacalin Leuven LE-435 5% BSA SavyonM121-0333. The prepared blots were placed in 90 mm petri dishes.4. The blots were blocked by adding to each petri dish 10 ml of anysuitable blocking solution well known to the skilled artisan (e.g. 5%bovine serine albumin).5. The dishes containing the blots in the blocking solution wereagitated gently by rotation on a rotating table (50 rpm) for 2 hours atroom temperature (or overnight at 4° C., without rotation).6. The blots were then washed by addition of 10 ml washing solution toeach petri dish. Any commonly available buffered solution (e.g.phosphate buffered saline) may be used for performing the washing steps.The dishes were washed by rotating gently (50 rpm) for 5 minutes. Theprocedure was performed a total of three times, discarding the oldwashing solution and replacing with fresh solution each time.

B: Addition of Milk Samples:

The milk samples used were as follows:

1. Bovine UHT long-life milk (3% fat) obtained from Ramat haGolandairies, Israel (lot 522104);2. Pasteurized goat's milk, obtained from Mechek dairies, Israel (lots 1and 2);3. Non-pasteurized goat's milked obtained as in 2. (lots 3 and 4).

The milk samples were diluted to 10% v/v and approximately 5 ml of eachsample applied to separate blots.

Duplicate blots were prepared for each of the aforementioned milksamples. In addition a further pair of blots were prepared without theaddition of saccharides (negative control).

The blots were then incubated at room temperature with agitation for onehour.

C. Colored Lectins:

From prior knowledge of the monosaccharide composition of the milkstested, and by application of a computer program based on the algorithmdescribed hereinbelow in Example 7, the following colored lectins werechosen: Con A, VVA.

A mixture of these two lectins was prepared in washing solution, suchthat the concentration of each colored lectin was 2 mg/ml.

500 μl of each lectin mix was incubated on the blots prepared asdescribed above. Each blot was read both by measuring the fluorescenceof fluorescein at 520 nm, and, in the case of the biotinylated lectin,measuring the signal of the TMB blue color produced following reactionof biotin with an HRP-streptavidin solution

The results obtained for the FITC-labeled and biotin-labeled lectins aregiven in Tables 12 and 13, respectively. The results presented in thesetables are measured on a 0 to 3 scale, wherein 0 represents a signalthat is below the noise level, and wherein results of 1-3 representpositive signals (above noise) following subtraction of the resultsobtained in the no-saccharide control.

Glycomolecule identity (GMID) cards obtained from these results forpasteurized goat's milk (lots 1 and 2), non-pasteurized goat's milk(lots 3 and 4) and bovine milk are shown in FIG. 1 (A to E,respectively). The positions of lectins 1 to 24 are shown in one rowfrom left to right at the top of each card 1.

D. Interpretation of Results:

The bovine milk sample yielded a GMID indicating that the polysaccharidein the sample contains saccharides that yield positive results forlectins specific for:

a. glucose/mannose (ConA, PSA and LCA);b. GlcNac (WGA and DSA).

The pasteurized goat milk samples yielded positive results for:

a. glucose/mannose (conA, PSA and LCA);b. GlcNac (DSA).

No difference in lectin reactivity between the lots tested was observed.

The non-pasteurized goat milk sample gave a positive reaction for:a. glucose/mannose (ConA, PSA and LCA);b. GlcNac (DSA).

In summary, the bovine milk differed from the goat's milk in that onlythe former reacted with WGA. There was essentially no difference betweenthe pasteurized and non-pasteurized goat's milk samples, with theexception that the signal intensity was significantly lower in thepasteurized samples.

Example 6 Glycomolecule Identity (GMID) Analysis of Lipopolysaccharides

A GMID analysis was performed on five different bacteriallipopolysaccharides obtained from Sigma Chemical Co. (St. Louis, Mo.,USA) (LPS#1, 7, 10, 15 and 16), essentially using the method asdescribed in Example 5, above. The colored lectins used were ECL, WGA,VVA and SBA.

The GMID cards obtained for samples LPS# 1, 7, 10, 15 and 16 are shownin FIG. 2 (A to E, respectively). It may be seen from this figure thatthe GMID cards provide unique “fingerprints” for each of the differentlipopolysaccharides, and may be used for identifying the presence ofthese compounds in samples containing bacteria or mixtures of theirproducts.

Example 7 Method for Selecting Colored Lectins

A number of factors must be taken into consideration when selectingcolored lectins for use in the method of polysaccharide analysisillustrated in Examples 5 and 6. Among these considerations are the needfor each of the chosen lectins to have a distinguishable color or otherdetectable marker, and for the need to reduce interactions betweenlectins. A flow chart illustrating an algorithm for use in coloredmarker selection is shown in FIG. 3. The algorithm shown in FIG. 3begins with the selection of n colored lectins (or other detectablemarkers) 101, said initial selection being made in accordance withinformation obtained about the partial or full monosaccharidecomposition of the saccharide to be analyzed.

In the next step 102, the colors of the selected lectins are examined inorder to check for identity/non-identity of the colors selected. Ifthere are identical colors in the selected group, then the processproceeds to step 103, otherwise the flow proceeds with step 104. In step103, one of the lectins that has been found to have a non-unique coloris replaced by another lectin that belongs to the same binding category(that is, one that has the same monosaccharide binding specificity); theflow proceeds to step 102.

In step 104, the n selected lectins are tested in order to detect anycross-reactivity with each other, and with the non-colored lectins usedin the first stage of the method described hereinabove in Example 5. Ifcross-reactivity is found, then the process continues to step 105,otherwise the flow proceeds to step 106, where the algorithm ends.

In step 105, one of the lectins determined to cross-react with anotherlectin is replaced by a lectin which does not cross-react; the flow thenproceeds to 102. The algorithm ends with step 106.

It is to be emphasized that while for values of n which are small, andfor saccharides with a simple monosaccharide composition, theabove-described algorithm may be applied by the operator himself/herselfmanually working through each step of the selection procedure.Alternatively (and especially for cases where n is a larger number orthe monosaccharide composition is more complex), the algorithmicprocesses described hereinabove may be performed by a computer programdesigned to execute said processes.

The above examples have demonstrated the usefulness of the methoddescribed herein. However, they have been added for the purpose ofillustration only. It is clear to the skilled person that manyvariations in the essentially sequence-specific agents used, in thereaction conditions therefor, in the technique of immobilization, and inthe sequence of labeling, reaction and detection steps may be effected,all without exceeding the scope of the invention.

Other Embodiments

The above Examples describe particular types of fingerprint assays andmethods according to the present invention. These assays may optionallybe performed with a variety of different configurations for “wet” orexperimental assay devices, hardware and software programs for gatheringand analyzing the data. FIG. 4 is a schematic block diagram of anexemplary method according to the present invention for performing afingerprint assay with the GMID card, which illustrates one type ofsystemic configuration and operation according to the present inventionfor performing the fingerprint assay. It should be noted that thisdescription is intended as an example only and is not meant to belimiting in any way.

As shown, in step 1, optionally and preferably, the saccharide-bindingagents are examined for efficacy before they are used in the assay withthe GMID card. In this example, the saccharide-binding agents aredescribed as lectins, although of course other such agents couldoptionally be used within the scope of the present invention. Morepreferably, each such lectin is examined for positive activity, mostpreferably through reactivity with a standard glycomolecule. Suchreactivity shows that the lectin is capable of binding to such astandard glycomolecule in a reproducible manner. Additionally and alsopreferably, the lectin should be tested for its ability to operate aseither saccharide-binding agent in the preferred embodiment of theassay, whether attached to the surface of the solid support, oralternatively present in a solubilized form.

In step 2, the lectins are optionally and preferably examined for theirability to bind to the solid support for the GMID card for theimmobilized saccharide-binding agent. In addition, optionally andpreferably, the solubilized form of the saccharide-binding agents isexamined in order to determine if there is any non-specific binding tothe solid support, which may increase levels of background lectinbinding, thereby degrading the signal of the specifically bound lectins.

In addition, more preferably the solid support for the GMID card isitself examined for various types of behaviors, such as generation ofbackground signals in the absence of specific lectin binding, and/orquenching of such signals. A particularly preferred solid support forthe GMID card of the present invention is a porous or semi-porousmembrane, such as nitrocellulose for example. Alternatively, the solidsupport could be a nitrocellulose coated solid surface such as a glassslide, for example, or any other suitable solid surface which has beencoated with a porous or semi-porous material.

In step 3, once the set of lectins has been selected for immobilizationon the solid support for the GMID card, and the support itself has alsobeen selected, then the GMID card, is prepared with the immobilizedlectins. Optionally, the GMID card may be prepared with “arrayer” or“spotting” devices, which are able to place relatively small, preciseamounts of lectins in a specific array on the solid support, to form anarray of a plurality of “spots”. These devices are also known as“microdispensing systems”, as they deposit volumes of material which aretypically measured in nanoliters, for example with an array of pins fordepositing such small volumes of material. Examples of suitable deviceswhich are operative with the present invention, include, but are notlimited to, Hydra™ (Robbins Inc., USA), MicroGrid II/TAS/Pro™(BioRobotics Ltd., United Kingdom) and GMS417™ (Genetic MicrosystemsAffymetrix Inc., USA).

Optionally and preferably, the lectins are pretreated before beingimmobilized to the solid surface or incubated with the GMID card in thesolubilized form. For example, such pretreatment could optionallyinclude periodation of the lectins in order to improve the signal tonoise ratio.

In step 4, optionally and more preferably, before being incubated withthe GMID card, the glycomolecules are treated to maximize the efficiencyof specific binding to the immobilized lectins on the support, and alsoto decrease non-specific binding to the immobilized lectins, the supportand the solubilized lectins. In addition, preferably the glycomoleculesare mixed with an appropriate buffer in order to form the samplesolution.

In step 5, the sample solution is contacted with the solid supportcontaining the immobilized lectins. Optionally, before the samplesolution is contacted with the solid support, the solid support iswashed with the sample buffer alone. The sample solution with theglycomolecules is then incubated with the solid support for anappropriate period of time. Optionally, a control solution is alsoincubated with at least a portion of the solid support, as a measurementfor non-specific binding.

In step 6, the solid support with the complexed glycomolecules is thenpreferably washed at least once with an appropriate washing buffer, aswell as with an appropriate blocking buffer. In step 7, the solidsupport is then incubated with the solubilized, labeled lectins as thesecond saccharide-binding agent. In step 8, again an appropriate washingprocedure is preferably performed.

In step 9, the signal from the labeled lectins is detected with anappropriate detection device. For example, if the label is chromogenic,then the detection device could be a CCD (charge-coupled device) camera.Clearly, one of ordinary skill in the art could select the appropriatedetection device according to the type of label on the lectin.

According to preferred embodiments of the present invention, the labelis a fluorescent dye, as previously described. For such a preferredembodiment, the detection device would also preferably include a lightsource of an appropriate wave length, for exciting the fluorescent dyelabel, and also an appropriate filter set for optionally filtering thelight from the light source and for filtering the resultant signal. Itshould be noted that such filters are not required for monochromaticlight sources, such as lasers for example. The possibility ofphotobleaching and the efficiency cofactor of each dye or fluorochromeis preferably considered in the analysis phase, as described in greaterdetail below.

The image of the entirety or at least a significant majority of the GMIDcard could optionally be obtained (as opposed to the detection of aplurality of single signals, for example). Examples of suitabledetection devices include “scanners” for obtaining at least a portion ofthe image of the GMID card, with multiple signals from a plurality of“spots”. Such devices may optionally be single band (light of a singlewavelength is detected); double band (light of two separate wavelengthsis detected); or spectrum devices (light is detected of at least two,but preferably a large number of, wavelengths).

In step 10, most preferably, the various signals from one or morecontrol “samples” are analyzed in order to determine the appropriatethreshold for the signal for the specifically bound lectins, as well asfor determining signal to noise ratios, and so forth. In addition, thesevarious signals can optionally be compared to the results from previousassays, in order to verify the quality of the assay for example.

In step 11, optionally and preferably the signals are examined in orderto determine the level of specific binding, if any, for example bysubtraction of background noise and by comparison to the threshold forspecifically bound lectins. The background noise is preferablydetermined as a function of the average noise, ±the standard deviation.

Steps 9-11 are optionally and preferably performed with a softwareprogram for controlling the process of capturing the signal, for examplein the form of image data; analyzing the control signals; and thenanalyzing the sample signals in order to obtain the actual assay data.Examples of suitable software programs include, but are not limited to,GeneTools™ (BioRobotics Ltd., United Kingdom); GenePix Pro 3.0™ (AxonInstruments Inc.) and QuantArray™ (GSI Lumonics Inc.). Alternatively,these steps could be optionally performed with firmware and/or hardware,or some combination thereof.

According to preferred embodiments of the present invention, these stepspreferably include the step of first defining the array for the “spots”.Such an array is optionally and more preferably defined automatically,and includes the definition of a grid for determining the expectedlocation of any specific signal from the “spots”. Next, the initiallocation of the spots is preferably determined in relation to the grid.Each individual spot is then centered, after which edge detection ispreferably performed to locate the boundary of each spot. Edge detectionis optionally performed according to a free form, determination of thesize and shape of the spots; a fixed form determination for the size andshape; or alternatively a fixed size but free shape determinationprocess. Any of these steps may be performed automatically oralternatively may be performed manually.

Next, the intensity of the signal for each spot is determined. Such anintensity is preferably determined relative to the background signal andto the signal to noise ratio, for example by subtracting the backgroundsignal from the raw signal data which is detected by the detectiondevice.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-47. (canceled)
 48. A method of diagnosing a disease in a subject, themethod comprising a) providing a substrate comprising at least one firstsaccharide-binding agent attached to a predetermined location on asurface of said substrate; b) contacting said substrate with acarbohydrate polymer from a biological sample from said subject underconditions allowing for the formation of a first complex between saidfirst saccharide-binding agent and said carbohydrate polymer; c)contacting said substrate with at least one second saccharide-bindingagent under conditions allowing for formation of a second complexbetween said first complex and said second saccharide-binding agent in asecond complex, wherein at least one of said first and secondsaccharide-binding agents comprises a plurality of differentsaccharide-binding agents; d) detecting said first saccharide-bindingagent and second saccharide-binding agent in said second complex; e)determining a fingerprint of binding of said first saccharide-bindingagent and said second saccharide-binding agent, wherein said fingerprintis a compilation of the binding status of the carbohydrate polymer andthe plurality of first saccharide-binding agents and secondsaccharide-binding agents, and correlating said fingerprint to adisease, thereby diagnosing said disease in said subject, wherein saiddisease is selected from the group consisting of a viral disease, anautoimmune disease, a bacterial infection and cancer.
 49. The method ofclaim 48, wherein said viral disease comprises HIV infection.
 50. Themethod of claim 48, wherein said autoimmune disease is insulin dependentdiabetes or rheumatoid arthritis.
 51. A method for diagnosing a diseasein a subject, the method comprising correlating a fingerprint of acarbohydrate polymer to a disease, wherein said disease is selected fromthe group consisting of a viral disease, an autoimmune disease, abacterial infection and cancer, wherein said fingerprint is obtained byproviding a carbohydrate polymer sample from said subject, contactingsaid carbohydrate polymer with a plurality of different essentiallysequence- and/or site-specific binding agents; determining whether saidcarbohydrate polymer binds to at least one of said plurality ofdifferent essentially sequence- and/or site-specific binding agents;contacting said carbohydrate polymer with a saccharide-binding agent,wherein at least one of the carbohydrate polymer sample orsaccharide-binding agent comprise a detectable label; detecting saidlabel; and determining whether said carbohydrate polymer binds to saidsaccharide-binding reagent, thereby generating said fingerprint fordiagnosing said disease.